Compositions and methods for enhancing triplex and nuclease-based gene editing

Abstract

Compositions for improved gene editing and methods of use thereof are disclosed. In a preferred method, gene editing involves use of a cell-penetrating anti-DNA antibody, such as 3E10, as a potentiating agent to enhance gene editing by nucleases and triplex forming oligonucleotides. Genomic modification occurs at a higher frequency when cells are contacted with the potentiating agent and nuclease or triplex forming oligonucleotide, as compared to the absence of the potentiating agent. The methods are suitable for both ex vivo and in vivo approaches to gene editing and are useful for treating a subject with a genetic disease or disorder. Nanoparticle compositions for intracellular delivery of the gene editing compositions are provided and are particularly advantageous for use with in vivo applications.

1 . A composition comprising
 a gene editing technology selected from the group consisting of triplex-forming molecules, pseudocomplementary oligonucleotides, a CRISPR system, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and intron encoded meganucleases, and   a potentiating agent that reduces one or more DNA repair pathways and increases genomic editing by the gene editing technology compared to the gene editing technology alone.   
     
     
         2 . The composition of  claim 1 , wherein the potentiating agent is a cell-penetrating antibody, fragment or humanized variant thereof. 
     
     
         3 . The composition of  claim 2 , wherein the cell-penetrating antibody is an anti-DNA antibody and inhibits RAD51. 
     
     
         4 . The composition of  claim 3 , wherein the cell-penetrating antibody comprises a 3E10 monoclonal antibody or a cell-penetrating fragment thereof; a monovalent, divalent, or multivalent single chain variable fragment (scFv); or a diabody; or humanized form or variant thereof. 
     
     
         5 . The composition of  claim 4 , comprising
 (i) the CDRs of any one of SEQ ID NO:1-6, 12, or 13 in combination with the CDRs of any one of SEQ ID NO:7-11, or 15;   (ii) first, second, and third heavy chain CDRs selected from SEQ ID NO [S]: 15-23 in combination with first, second and third light chain CDRs selected from SEQ ID NO [S]: 24-30;   (iii) a humanized forms of (i) or (ii);   (iv) a heavy chain comprising an amino acid sequence comprising at least 85% sequence identity to any one of SEQ ID NO:1 or 2 in combination with a light chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:7 or 8;   (v) a humanized form of (iv); or   (vi) a heavy chain comprising an amino acid sequence comprising at least 85% sequence identity to any one of SEQ ID NO:3-6 in combination with a light chain comprising an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO:9-11.   
     
     
         6 - 9 . (canceled) 
     
     
         10 . The composition of  claim 1 , further comprising a donor oligonucleotide that induces a mutation(s) in the cell's genome by insertion or recombination induced or enhanced by the gene editing technology. 
     
     
         11 - 19 . (canceled) 
     
     
         20 . A pharmaceutical composition comprising the composition of  claim 1  and a pharmaceutically acceptable excipient. 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . A method of modifying the genome of a cell comprising contacting the cell with a gene editing technology selected from the group consisting of triplex-forming molecules, pseudocomplementary oligonucleotides, a CRISPR system, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and intron encoded meganucleases, and
 a potentiating agent that reduces one or more DNA repair pathways and increases genomic editing by the gene editing technology compared to the gene editing technology alone.   
     
     
         24 . The method of  claim 23 , wherein the gene editing technology and potentiating agent are part of different compositions. 
     
     
         25 - 34 . (canceled) 
     
     
         35 . The method of  claim 23 , wherein the cell's genome has a mutation underlying a disease or disorder selected from the group consisting of hemophilia, muscular dystrophy, globinopathies, cystic fibrosis, xeroderma pigmentosum, and lysosomal storage diseases, immune deficiency syndromes such as X-linked severe combined immunodeficiency and ADA deficiency, tyrosinemia, Fanconi anemia, the red cell disorder spherocytosis, alpha-1-anti-trypsin deficiency, Wilson's disease, Leber's hereditary optic neuropathy, and chronic granulomatous disorder. 
     
     
         36 . The method of  claim 35 , wherein the mutation is in a gene encoding coagulation factor VIII, coagulation factor IX, dystrophin, beta-globin, CFTR, XPC, XPD, DNA polymerase eta, Fanconi anemia genes A through L, SPTA1 and other spectrin genes, ANK1 gene, SERPINA1 gene, ATP7B gene, interleukin 2 receptor gamma (IL2RG) gene, ADA gene, FAH gene, and genes linked to chronic granulomatous disease including the CYBA, CYBB, NCF1, NCF2, or NCF4 genes. 
     
     
         37 . (canceled) 
     
     
         38 . The method of  claim 23 , wherein the contacting occurs ex vivo. 
     
     
         39 . The method of  claim 38 , wherein the cell is a hematopoietic stem cell. 
     
     
         40 . The method of  claim 23 , further comprising administering a plurality of the cells to a subject in need thereof. 
     
     
         41 . The method of  claim 40 , wherein the cells are administered to the subject in an effective amount to treat one or more symptoms of a disease or disorder. 
     
     
         42 . The method of  claim 23 , wherein the contacting occurs in vivo following administration to a subject in need thereof. 
     
     
         43 . The method of  claim 42 , wherein the subject has a disease or disorder selected from the group consisting of hemophilia, muscular dystrophy, globinopathies, cystic fibrosis, xeroderma pigmentosum, and lysosomal storage diseases, immune deficiency syndromes such as X-linked severe combined immunodeficiency and ADA deficiency, tyrosinemia, Fanconi anemia, the red cell disorder spherocytosis, alpha-1-anti-trypsin deficiency, Wilson's disease, Leber's hereditary optic neuropathy, and chronic granulomatous disorder. 
     
     
         44 . The method of  claim 43 , wherein gene modification occurs in an effective amount to reduce one or more symptoms of the disease or disorder in the subject. 
     
     
         45 . The method of  claim 23 , wherein the gene editing technology, potentiating agent, and optional donor oligonucleotide are encapsulated in nanoparticles together or separately. 
     
     
         46 . (canceled) 
     
     
         47 . (canceled) 
     
     
         48 . The method of  claim 45 , wherein a targeting moiety, a cell penetrating peptide, or a combination thereof is associated with, linked, conjugated, or otherwise attached directly or indirectly to the nanoparticle. 
     
     
         49 - 54 . (canceled)